JP5535920B2 - RNA vaccine - Google Patents

Description

Detailed Description of the Invention

  The present invention relates to RNA vaccines.

  In recent decades, type I allergic diseases have emerged as a major health problem in developed countries in Europe and the United States, and now about 25% of the population has developed.

  In addition to genetic factors, growth factors, including infections in early childhood, dietary habits, and environmental factors such as exposure to passive smoking and air pollution are greatly related to the occurrence of atopic diseases.

  Currently, specific immunotherapy, in which allergens are given incrementally over the years, is the only effective therapeutic intervention. However, because of the high dose, the risk of anaphylactic side effects is obvious and can cause patient sensitization to unidentified components when using unpurified, less-identified allergen extracts There is.

  Furthermore, vaccination against type I allergy has not been realized, and the most feasible approach would be to prevent the development of allergic disease in infants who have a high genetic risk of developing allergic disease. It is easier to train a young immune system than to balance the already developed allergic immune phenotype.

  Ying et al. (Nature Med (1999) 5: 823-827) describes a self-repairing RNA vaccine containing RNA encoding β-galactosidase often used as a model molecule for studying immunological processes. Is disclosed. In Ying et al., Anti-tumor responses have been studied and the induction of CD8 positive cells has been observed. However, CD4 positive cells not investigated in Ying et al., In contrast to CD8 positive cells, mediate immunological protection against allergies and prevent class switching to IgE in B cells.

  In recent years, nucleic acid-based vaccines have become an effective technique for biasing the immune mechanism underlying allergic diseases. Numerous animal experiments have demonstrated that DNA vaccines suppress the induction of type I allergic reactions and even restore existing allergic TH2 immune status (Weiss, R. et al. (2006) Int Arch Allergy Immunol 139: 332-345).

  Nevertheless, concerns have been raised regarding the safety of DNA-based vaccines. Injected DNA molecules can be integrated into the host genome, and due to dispersibility in various tissues, allergens are released continuously, resulting in patients with existing allergen-specific IgE molecules There are considerations in inducing an uncontrollable anaphylactic reaction. Furthermore, when administering vaccines to healthy children, it is necessary to apply the highest standards of safety in anti-allergic vaccines.

  Accordingly, an object of the present invention is to provide an allergen vaccine that overcomes the drawbacks of DNA vaccines and is effective in treating allergy or that can successfully suppress sensitization to allergens.

  The present invention relates to an RNA vaccine comprising at least one RNA molecule encoding at least one allergen or a derivative of the allergen. In the RNA vaccine, the allergens are Alnus glutinosa, Alternaria alternata, Ambrosia artemisiifolia, Apium graveolens, Arakis hypogaea (Arachis hypogaea) , Betula verrucosa, Carpinus betulus, Castanea sativa, Cladosporiumherbarum, Corylus avellana, Cryptomeria yp tom , Cyprinus carpio, Daucus carota, Dermatophagoides pteronyssinus, Fagus sylvatica, Fu Of squirrel domesticus (Felis domesticus), Hevea brasiliensis (Heveabrasiliensis), Juniperus ashei, Malus domestica, Malus domestica, Quercus alba, and Phleum pratense It is an allergen.

  It has been found that an RNA molecule encoding an allergen or a derivative of the allergen may be effectively used as an RNA vaccine. RNA vaccines are characterized by DNA vaccines for the treatment of allergic diseases. That is, in RNA vaccines, allergens are provided in their purest form, ie, genetic information, and like DNA vaccines, RNA vaccines elicit TH1-biased immune responses. In addition, methods similar to those made for DNA vaccines to produce hypoallergenic gene products can be used in RNA vaccines.

Furthermore, RNA vaccines have significant advantages over DNA vaccines.
(I) RNA vaccines contain pure genetic information of allergens, but are foreign, such as viral promoters, antibiotic resistance genes, or viral / bacterial regulatory sequences normally present in the backbone of plasmids used in DNA vaccines Does not contain an array.
(Ii) Since RNA is not integrated into the host genome, the risk of malignant tumors can be eliminated.
(Iii) Since RNA is translated in the cytoplasm of the cell, there is no need for a transcriptional mechanism of the cell nucleus, and RNA vaccines are independent of nuclear translocation or nuclear translocation, and independent of each nuclear stage. doing.
(Iv) Since RNA is rapidly degraded, foreign genes are expressed in a short period of time, and it is possible to avoid long-term uncontrollable antigen expression.

  The RNA vaccine according to the present invention may contain one or more RNA molecules encoding allergens, and may preferably contain 2, 3, 5, 10 kinds of RNA molecules. However, one RNA molecule may encode one or more allergens. That is, one RNA molecule may include a base sequence that encodes at least 1, 2, 3, 5, 10 different or the same allergen. The allergen encoded by one or more RNA molecules may be selected from any combination of the enumerations described below.

  As used herein, the term “RNA vaccine” refers to a vaccine comprising an RNA molecule as defined herein. It will be appreciated, however, that the vaccine may contain other substances and molecules (eg pharmaceutical excipients) that are required or suitable for administration to an individual.

  The term “allergen of” can be replaced by the terms “allergen derived from” and “allergen derived from”. This means that the allergen is naturally expressed in the organism and that DNA / RNA encoding the allergen is isolated to produce the RNA molecule of the present invention.

  It has been found that not all RNA molecules encoding allergens induce the formation of allergen-specific antibodies when administered to mammals or humans. For example, RNA molecules encoding Artemisia vulgaris allergen Art V1 and Olea europea allergen Ole e 1 are unable to induce Th1 memory and induce allergen-specific IgE responses. It cannot be suppressed. However, RNA molecules encoding allergens derived from the sources described above can do the above.

  According to a preferred embodiment of the present invention, the allergen of Arnus glutinosa is Aln g 1, and the allergen of Alternaria alternata is Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, The allergens of Ambrosia artemisifolia selected from the group consisting of Alt a 7, Alt a 8, Alt a 10, Alt a 12, and Alt a 13 are Amb a 1, Amb a 2, Amb a 3, Amb a 5 , Amb a 6, Amb a 7, Amb a 8, Amb a 9 and Amb a 10, and the allergen of Apium gravelens is selected from the group consisting of Api g 1, Api g 4 and Api g 5 Arakis Hypogaea Allerge Is selected from the group consisting of Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7 and Ara h 8, and the allergen of Betula velocosa is Bet v 1 , Bet v 2, Bet v 3, Bet v 4, Bet v 6 and Bet v 7, the allergen of Carpinus betulus is Car b 1, and the allergen of Castanea sativa is Cas s 1 , Cas s 5 and Cas s 8 and the allergens of Cladosporium herbalum are Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9, Cla h Selected from the group consisting of 10 and Cla h 12, The allergen is selected from the group consisting of Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, and Cor a 11, and the Cryptomeria japonica allergen consists of Cry j 1 and Cry j 2. The allergen of Caprinus carpio selected from the group is Cyp c 1, the allergen of Daux Carota is selected from the group consisting of Dau c 1 and Dau c 4, and the allergen of Dermatophagoides pteronisinus is Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8, Der p 9, Der p 10, Der p 11, Der p 14, Der p 20 , Der p 21 and Clone 30 And the allergen of Fagus Silvatica is Fags 1 and the allergen of Ferris domesticus is Feld1, Feld2, Feld3, Feld4, Feld5w, Feld d Selected from the group consisting of 6w and Fel d 7w, Hevea brushriensis allergens are Hev b 1, Hev b 2, Hev b 3, Heb b 4, Hev b 5, Hev b 6.01, Hev b 6 .02, Hev b 6.03, Hev b 7.01, Hev b 7.02, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12 and Hev b 13 The allergens of Junipels Assure are Jun a 1 and Jun a 2. And a mars domestica allergen selected from the group consisting of Mal d 1, Mal d 2, Mal d 3 and Mal d 4, and an allergen of Kerks alba is que a 1 And the allergen of Fleum platen is the group consisting of Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12, and Phl p 13 More selected.

According to a preferred embodiment of the invention, the allergen is selected from the group consisting of:
Grass pollen: Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 7, Phl p 12
House dust mite: Der p 1, Der p 2, Der p 7, Der p 21, Clone 30 allergen (PCT international application number AT2007 / 000201, Austrian patent application number AT 503530:
MKFNIIIVFI SLAILVHSSY AANDNDDDPT TTVHPTTTEQ PDDKFECPSR FGYFADPKDP HKFYICSNWE AVHKDCPGNT RWNEDEETCT, SEQ ID NO: 1)
Birch pollen: Bet v 1 and its similar tree (Aln g 1, Cor a 1, Fag s 1) or food allergen (Mal d 1, Api g 1, Pru p 1)
Cat: Fel d 1, Fel d 2
Grass (Ragweed, Artemisia): Amb a 1
Cypress / juniper / cedar: Cry j 1, Cry j 2, Jun a 1, Jun a 3,
Cha o 1, Cha o 2, Cup a 1, Cup a 3, Jun a 1, Jun a 3, Pla a 3
Peanuts: Ara h 1, Ara h 2, Ara h 4
Hazelnut: Cor a 8, Cor a 9
Fish / shrimp: Gad c 1, Cyp c 1, Pen a 1.

  Particularly suitable allergens used in the RNA vaccine according to the invention are Aln g 1, Alt a 1, Amb a 1, Apig 1, Ara h 2, Bet v 1, beta-casein, Car b 1, Cas s 1, Cla h 8, Cor a 1, Cry j 1, Cyp c 1, Dau c 1, Der p 2, Fag s 1, Feld d 1, Heb b 6, Jun a 1, Mal d 1, ovalbumin (OVA) ), Phl p 1, Phl p 2, Phl p 5, Phl p 6, and Phl p 7.

  The allergens described above have been found to be particularly suitable for use in RNA vaccines. Naturally, Amb a 1, Amb a 2, Amb a 3, Amb a 5, Amb a 6, Amb a 7, Amb a 8, Amb a 9, Amb a 10, Amb t 5, Hel a 1, Hel a 2, Hel a 3, Mer a 1, Che a 1, Che a 2, Che a 3, Sal k 1, Cat r 1, Pla l 1, Hum j 1, Par j 1, Par j 2, Par j 3 , Par o 1, Cyn d 1, Cyn d 7, Cyn d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24, Dac g 1, Dac g 2, Dac g 3, Dac g 5, Fes p 4w, Hol l 1, Lol p 1, Lol p 2, Lol p 3, Lol p 5, Lol p 11, Pha a 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6 , Phl p 11, Phl p 12, Phl p 13, Poa p 1, Poa p 5, Sor h 1, Pho d 2, Aln g 1, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v 7, Car b 1, Cas s 1, Cas s 5, Cas s 8, Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Que a 1 , Fra e 1, Lig v 1, Syr v 1, Cry j 1, Cry j 2, Cup a 1, Cup s 1, Cup s 3w, Jun a 1, Jun a 2, Jun a 3, Jun o 4, Jun s 1, Jun v 1, Pla a 1, Pla a 2, Pla a 3, Aca s 13, Blot 1, Blot 3, Blot 4, Blot 5, Blot 6, Blot 10, Blot 11 , Blo t 12, Blo t 13, Blo t 19, Der f 1, Der f 2, Der f 3, Der f 7 , Der f 10, Der f 11, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18w, Der m 1, Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8, Der p 9, Der p 10, Der p 11, Der p 14, Der p 20, Der p 21, Eur m 2, Eur m 14, Gly d 2 , Lep d 1, Lep d 2, Lep d 5, Lep d 7, Lep d 10, Lep d 13, Tyr p 2, Tyr p 13, Bos d 2, Bos d 3, Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Can f 1, Can f 2, Can f 3, Can f 4, Equ c 1, Equ c 2, Equ c 3, Equ c 4, Equ c 5, Fel d 1 , Fel d 2, Fel d 3, Fel d 4, Fel d 5w, Fel d 6w, Fel d 7w, Cav p 1, Cav p 2, Mus m 1, Rat n 1, Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8, Alt a 10, Alt a 12, Alt a 13, Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8 , Cla h 9, Cla h 10, Cla h 12, Asp fl 13, Asp f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 5, Asp f 6, Asp f 7, Asp f 8, Asp f 9, Asp f 10, Asp f 11, Asp f 12, Asp f 13, Asp f 15, Asp f 16, Asp f 17, Asp f 18, Asp f 22w, Asp f 23, Asp f 27, Asp f 28 , Asp f 29, Asp n 14, Asp n 18, Asp n 25, Asp o 13, Asp o 21, Pen b 13, Pen b 26, Pen ch 13, Pen ch 18, Pen ch 20, Pen c 3, Pen c 13, Pen c 19, Pen c 22w, Pen c 24, Pen o 18, Fus c 1, Fus c 2, Tri r 2, Tri r 4, Tri t 1, Tri t 4, Cand a 1, Cand a 3, Cand b 2, Psi c 1, Psi c 2, Cop c 1, Cop c 2, Cop c 3, Cop c 5, Cop c 7, Rho m 1, Rho m 2, Mala f 2, Mala f 3, Mala f 4, Mala s 1, Mala s 5, Mala s 6, Mala s 7, Mala s 8, Mala s 9, Mala s 10, Mala s 11, Mala s 12, Mala s 13, Epi p 1, Aed a 1, Aed a 2, Api m 1, Api m 2, Api m 4, Api m 6, Api m 7, Bom p 1, Bom p 4, Bla g 1, Bla g 2, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Per a 1, Per a 3, Per a 6, Per a 7, Chi k 10, Chi t 1-9, Chi t 1.01, Chi t 1.02, Chi t 2.0101, Chi t 2.0102, Chi t 3, Chi t 4, Chi t 5, Chi t 6.01, Chi t 6.02, Chi t 7, Chi t 8, Chi t 9, Cte f 1, Cte f 2, Cte f 3, Tha p 1, Lep s 1, Dol m 1, Dol m 2, Dol m 5, Dol a 5, Pol a 1, Pol a 2, Pol a 5, Pol d 1, Pol d 4, Pol d 5, Pol e 1, Pol e 5, Pol f 5, Pol g 5, Pol m 5, Vesp c 1, Vesp c 5, Vesp m 1, Vesp m 5, Ves f 5, Ves g 5, Ves m 1, V es m 2, Ves m 5, Ves p 5, Ves s 5, Ves vi 5, Ves v 1, Ves v 2, Ves v 5, Myr p 1, Myr p 2, Sol g 2, Sol g 4, Sol i 2, Sol i 3, Sol i 4, Sol s 2, Tria p 1, Gad c 1, Sal s 1, Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d 5, Met e 1, Pen a 1, Pen i 1, Pen m 1, Pen m 2, Tod p 1, Hel as 1, Hal m 1, Ran e 1, Ran e 2, Bra j 1, Bra n 1, Bra o 3, Bra r 1, Bra r 2, Hor v 15, Hor v 16, Hor v 17, Hor v 21, Sec c 20, Tri a 18, Tri a 19, Tri a 25, Tri a 26, Zea m 14, Zea m 25, Ory s 1, Api g 1, Api g 4, Api g 5, Dau c 1, Dau c 4, Cor a 1.04, Cor a 2, Cor a 8, Fra a 3, Fra a 4, Mal d 1, Mal d 2, Mal d 3, Mal d 4, Pyr c 1, Pyr c 4, Pyr c 5, Pers a 1, Pru ar 1, Pru ar 3, Pru av 1, Pru av 2, Pru av 3, Pru av 4, Pru d 3, Pru du 4, Pru p 3, Pru p 4, Aspa o 1, Cros 1, Cros 2, Lac s 1, Vit v 1, Mus xp 1, Ana c 1, Ana c 2, Cit l 3, Cit s 1, Cit s 2, Cit s 3, Lit c 1, Sin a 1, Gly m 1, Gly m 2, Gly m 3, Gly m 4, Vig r 1, Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ar ah 6, Ara h 7, Ara h 8, Len c 1, Len c 2, Pis s 1, Pis s 2, Act c 1, Act c 2, Cap a 1w, Cap a 2, Lyc e 1, Lyc e 2 , Lyc e 3, Sola t 1, Sola t 2, Sola t 3, Sola t 4, Ber e 1, Ber e 2, Jug n 1, Jug n 2, Jug r 1, Jug r 2, Jug r 3, Ana o 1, Ana o 2, Ana o 3, Ric c 1, Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, Cuc m 1, Cuc m 2, Cuc m 3 , Ziz m 1, Ani s 1, Ani s 2, Ani s 3, Ani s 4, Arg r, Asc s 1, Car p 1, Den n 1, Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6.01, Hev b 6.02, Hev b 6.03, Hev b 7.01, Hev b 7.02, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13, Other allergens such as Hom s 1, Hom s 2, Hom s 3, Hom s 4, Hom s 5, and Trip s 1 can also be used in the present invention.

  According to a preferred embodiment according to the invention, the allergen derivative is hypoallergenic.

  In order to elicit a specific immune response without inducing or minimizing an allergic reaction in a mammal, particularly a human body, the allergen of the present invention or a derivative thereof exhibits hypoallergenicity, That is, the hypoallergenic molecule preferably exhibits no or little IgE reaction.

  As used herein, the term “hypoallergenic” refers to a T, specific for an allergen when a peptide, polypeptide or protein derived from an allergen with allergic properties is administered to an individual. It refers to the ability to induce cell induction and have little or no allergic reaction. By preserving T cell epitopes present in the allergen and removing or destroying IgE-binding epitopes from the allergen, the ability to induce a human allergic reaction can be eliminated or reduced in hypoallergenic derivatives of the allergen . For example, it can be achieved by splitting into fragments that have reduced or no IgE binding capacity. Some or all of the divided fragments may be combined in an order different from the order of the fragments in the wild-type allergen. (See, for example, EP 14409779). Another method for generating hypoallergenic molecules from allergens is a method involving a C-terminal deletion and / or an N-terminal deletion of a wild-type allergen (see, for example, European Patent Application No. 1224215). Of course, hypoallergenic molecules can also be generated by introducing specific mutations into one or more amino acid residues of the wild type allergen. As a result of this modification, the three-dimensional structure is deleted.

  Hypoallergenicity of RNA vaccines is achieved by targeting proteins produced from RNA vaccines to the ubiquitination pathway of cells and degrading each protein into hypoallergenic peptides in the ubiquitination pathway. The above is accomplished by attaching a sequence encoding ubiquitin to the 5 'end of the RNA encoding the allergen. The ubiquitination efficiency can be increased by mutating the 76th amino acid residue from glycine to alanine (G76 → A76). Moreover, the ubiquitination efficiency can be further increased by mutating the first amino acid (methionine) of the allergen to a destabilizing amino acid (arginine) (M77 → R77). The gene product resulting from the RNA vaccine can also be ubiquitinated by adding a carboxy terminal destabilizing sequence known as the PEST sequence.

  According to a preferred embodiment of the invention, the IgE reactivity of the hypoallergenic allergen derivative encoded by the RNA in the vaccine is 10% or more, preferably 20% or more, than the IgE reactivity of the wild type allergen, More preferably, it is 30% or more, particularly 50% or more.

  By translating RNA in vitro in the rabbit reticulocyte lysate system, the hypoallergenicity of the RNA vaccine can be periodically tested. Gene products generated from RNA vaccines can be analyzed by IgE Western blotting using a pool of appropriate patient sera. The IgE binding ability of each hypoallergenic allergen can be evaluated as compared to the IgE binding ability of the wild-type molecule translated in the reticulocyte lysate system.

  According to a particularly preferred embodiment according to the invention, the RNA molecule of the invention has more than 1, preferably more than 2, more preferably more than 3, more preferably more than 4 allergens. Alternatively, it may encode a derivative of the allergen. In particular, the RNA molecules encode Phl p 1, Phl p 2, Phl p 5 and Phl p 6, or Alng 1, Cor a 1, Quea 1, Car b 1 and Bet v 1.

  The RNA molecule encoding the allergen or derivative thereof is bound to at least one additional peptide, polypeptide, or protein.

  An RNA sequence encoding an allergen can be linked to an RNA sequence encoding a peptide, polypeptide or protein. These peptides may be signal peptides, such as human tissue plasminogen activation signal peptide (hTPA), that enhance protein secretion from cells by directing allergens to the endoplasmic reticulum. The peptide or protein may be a membrane protein (LAMP) bound to lysosomes or the C-terminal 20-amino acid in lysosomal integral membrane protein-II (LIMP-II). The LAMP / LIMP-II sequence is used to direct antigenic proteins to the major histocompatibility class II (MHC II) vesicle compartment of transformed antigen presenting cells (APCs), thereby accelerating vaccine efficacy Increase the activity of T helper cells. The protein or polypeptide may be a protein that increases the TH bias of the vaccine, such as heat shock protein 70 (HSP70), bacterial toxins such as cholera toxin, or heat-labile enterotoxin (LT) of Escherichia coli. .

  In a preferred embodiment according to the present invention, the RNA molecule comprises at least one further element selected from the group consisting of replicase, β-globin leader sequence, cap0, cap1 and poly A tail.

  The RNA vaccine of the present invention is constituted by an RNA sequence encoding each allergen. The RNA sequence may be an allergen wild-type sequence or adapted to its codon usage. Adapting codon usage can increase the translation efficiency and half-life of RNA. A poly A tail consisting of at least 30 adenosine residues is attached to the 3 ′ end of the RNA to increase the half-life of the RNA. The 5 'end of RNA is capped with a mutant ribonucleotide having m7G (5') ppp (5 ') N (cap 0 structure) structure or a derivative thereof. The mutated ribonucleotide or derivative thereof may be incorporated during RNA synthesis or after RNA transcription from Vaccinia Virus Capping Enzyme (VCE: mRNA triphosphatase, guanylyltransferase and guanine-7-methyltransferase). The enzyme treatment may be performed using an enzyme. This enzyme catalyzes the composition of the N7-monomethylated cap 0 structure. The cap 0 structure plays an important role in maintaining the stability and translation efficiency of RNA vaccines. The 5 'cap of the RNA vaccine may be further modified by 2'-O-methyltransferase, which generates a cap 1 structure (m7Gppp [m2'-O] N) and further improves translation efficiency.

  RNA vaccines can be optimized by conversion to self-replicating vaccines. The vector contains, together with the gene of interest, an alphavirus-derived replication element and a structural viral protein surrogate. RNA vaccines based on replicase have been shown to elicit not only antibodies but also cytotoxic responses, even at very low doses, due to immune activation mediated by virus-derived danger signals (Ying, H. et al. (1999) Nat Med 5: 823-827).

  The RNA vaccine may also be a self-replicating RNA vaccine. Self-replicating RNA vaccines are derived from Semliki Forest fever virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalomyelitis virus (VEE), Ross River virus (RRV), or other viruses belonging to the alphavirus family It is composed of replicase RNA molecules. Downstream of the replicase is a subgenomic promoter that controls the replication of the allergen RNA followed by an artificial poly A tail composed of 30 or more adenosine residues.

  According to another preferred embodiment of the invention, the vaccine further comprises CpG-DNA and a cytokine, which is preferably interleukin (IL) -12 and IL-15.

  Furthermore, the vaccine or the vaccine formulation according to the invention may contain an adjuvant. An “adjuvant” in the present invention refers to a compound or mixture that enhances the immune response to an antigen. The adjuvant may be supplied as a tissue sustained-release preparation for sustained release of the antigen. Adjuvants include, among others, complete Freund's adjuvant, incomplete Freund's adjuvant, mineral gels such as saponin and aluminum hydroxide, surfactants such as lysolecithin, pluronic polyhydric alcohols, polyvalent anions, peptides, levamisole, CpG-DNA, Contains oil or hydrocarbon emulsions and potentially beneficial adjuvants such as BCG (Bacille Calmette Guerin) and Corynebacterium parvum.

  Alternatively or in addition to the above, immunostimulatory proteins may be used as adjuvants or for increased immunity against the vaccine. Co-administration of immunostimulatory molecules such as immunostimulatory cytokines, immune enhancing cytokines or inflammatory cytokines, lymphokines, or chemokines with vaccines, particularly vector vaccines, may increase the effectiveness of vaccination (Salgaller and Lodge, J. Surg. Oncol. (1988) 68: 122). For example, IL-2, IL-3, IL-12, IL-15, IL-18, IFN-gamma, IL-10, TGF-beta, granulocyte macrophage (GM) -colony stimulating factor (CSF) and others Colony stimulating factor, macrophage inflammatory factor, Flt3 ligand (Lyman, Curr. Opin. Hematol., 1998, 5: 192), CD40 ligand, and other cytokines or cytokine genes, as well as some major costimulatory molecules or Those genes (eg, B7.1, B7.2) can be used. These immunostimulatory molecules may be distributed systemically or locally as proteins, or may be encoded by RNA molecules or RNA molecules in the RNA vaccine according to the present invention. Moreover, you may use polycationic peptides, such as polyarginine, as an immunostimulatory molecule.

  According to a further preferred embodiment according to the invention, the vaccine is applied for intramuscular administration, intradermal administration, intravenous administration, transdermal administration, topical administration, or particle gun administration.

  Various methods may be used to administer the RNA vaccine according to the present invention. For example, in one method, the RNA vaccine is injected directly into the body in vivo (eg, intramuscular, intradermal, intravenous, intranasal, etc.). In addition, RNA can be injected into cells (eg, epithelial cells) outside the body. For example, RNA vaccines are transfected into epithelial cells in vitro and administered (transplanted) to the body. If exogenous RNA or heterologous RNA is injected into the cell, the cell can be transfected with the RNA. RNA can be introduced into cells by pulsing, ie, culturing the cells with the RNA molecules of the present invention. Alternatively, RNA may be introduced in vivo by lipofection as naked RNA or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids are used to generate liposomes for in vivo transfection. Lipid compounds and compositions useful for nucleic acid transfection are, for example, DODC, DOPE, CHOLU, DMEDA, DDAB, DODAC, DOTAP and DOTMA. Other molecules are also useful in facilitating transfection of nucleic acids in vivo. Other molecules include cationic oligopeptides (for example, WO 95/21931), peptides derived from DNA-binding proteins (for example, WO 96/25508), or cationic polymers (for example, WO 95/21508). / 21931 pamphlet). Polyethyleneimine and its derivatives, polylactide-polyglycolide and chitosan may also be used. Alternatively, RNA molecules can be introduced into desired host cells using well-known methods such as electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, or gene gun. (Refer to fine particle gun transfection, Tang et al., Nature (1992) 356: 152-154).

  Another aspect of the invention relates to the use of at least one RNA molecule for the manufacture of a vaccine for the treatment or prevention of allergies as defined herein.

  A further aspect of the invention relates to the use of at least one RNA molecule for producing a vaccine for desensitization of an individual to an allergen as defined herein.

  According to another preferred embodiment of the present invention, the vaccine is applied for intramuscular administration, intradermal administration, intravenous administration, transdermal administration or particle gun administration.

  Another aspect according to the invention relates to an isolated RNA molecule comprising at least one nucleotide encoding at least one allergen or derivative thereof. The RNA molecule is preferably at least one base selected from the group consisting of cap0, cap1, 5′β-globin leader sequence, self-replicating RNA, recoded allergen sequence, and artificial poly A tail. Contains an array. Among them, the RNA molecule is particularly preferably cap0-allergen sequence-polyA tail. cap0 is beneficial for the generation of antibodies in vivo and for self-replicating RNA vaccines for allergen-specific T cell induction and IFN-gamma secretion.

  Furthermore, although this invention is demonstrated using the following drawings and examples, this invention is not limited to them.

  FIG. 1 shows transfection of BHK21 cells in vitro with RNA encoding β-galactosidase (βGal-RNA) or self-replicating RNA (βGal-repRNA) transcripts. RNA transcripts (cap) with or without the m7G (5 ') ppp (5') G cap structure were tested (no cap). Untransfected cells were used as a background control (not transfected). Data are shown as the mean ± standard error (SEM) of three independent transfection experiments.

  FIG. 2A shows Phl p 5-specific IgG1 and IgG2a levels after nucleic acid vaccination, and FIG. 2B shows subsequent sensitization with recombinant allergens in alum. Serum was diluted 1: 1000 (FIG. 2A), 1: 100,000 (FIG. 2B). The numerical value on the top of the bar graph indicates the average ratio of IgG1: IgG2 in each group. Data represent mean ± SEM (n = 4).

  FIG. 3 shows Phl p 5 specific IgE measured by RBL release assay. IgE levels were measured after vaccination with each nucleic acid vaccine (grey bar graph) and after subsequent sensitization with recombinant allergen in alum (black bar graph). Numbers represent the mean ± SEM (n = 4) of specific hexosaminidase release. ***: P <0.001.

FIG. 4 shows the number of IFN-gamma (FIG. 4A), IL-4 (FIG. 4B), and IL-5 (FIG. 4C) after in vitro restimulation with recombinant Phl P5, measured using ELISPOT. Indicates. Data show mean ± SEM (n = 4) of the number of cytokine secreting cells per 10 ⁶ splenocytes.

  FIG. 5 shows the total white blood cell count (FIG. 5A) and eosinophil count (FIG. 5B) in BALF of mice sensitized after intranasal administration of allergen. A numerical value shows average value +/- SEM (n = 4). *: P <0.05; **: P <0.01.

  FIG. 6 shows IL-5 (FIG. 6A) and IFN-γ (FIG. 6B) levels in BALF of sensitized mice following intranasal administration of allergens. Data show mean ± SEM (n = 4). *: P <0.05; **: P <0.01; ***: P <0.001.

  FIG. 7 shows the induction of Th1 memory by RNA pTNT-Bet v1 and the suppression of the IgE response.

  FIG. 8 shows the induction of Th1 memory and suppression of IgE response by RNA pTNT-Car b1.

  FIG. 9 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Cas s1.

  FIG. 10 shows the induction of Th1 memory by RNA pTNT-Phl p1.

  FIG. 11 shows the induction of Th1 memory by RNA pTNT-Phl p6 and the suppression of the IgE response.

  FIG. 12 shows the induction of Th1 memory by RNA pTNT-Cor a1.

  FIG. 13 shows the induction of Th1 memory by RNA pTNT-Aln g1.

  FIG. 14 shows the induction of Th1 memory by RNA pTNT-Fag s1 and the suppression of IgE response.

  FIG. 15 shows the induction of Th1 memory by RNA pTNT-Phl p2 and the suppression of IgE response.

  FIG. 16 shows the induction of Th1 memory by RNA pTNT-Phl p7 and the suppression of the IgE response.

  FIG. 17 shows the induction of Th1 memory by RNA pTNT-hybrid (Phl p1-2-5-6) and the suppression of IgE response.

  FIG. 18 shows the induction of Th1 memory by RNA pTNT-Cry j1.

  FIG. 19 shows the induction of Th1 memory by RNA pTNT-Jun a1.

  FIG. 20 shows the induction of Th1 memory by RNA pTNT-Amb a1.

  FIG. 21 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Api g1.

  FIG. 22 shows the induction of Th1 memory by RNA pTNT-Dau c1.

  FIG. 23 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Mal d1.

  FIG. 24 shows the induction of Th1 memory by RNA pTNT-Ova and the suppression of the IgE response.

  FIG. 25 shows the induction of Th1 memory by RNA pTNT-Beta-Casein and the suppression of the IgE response.

  FIG. 26 shows the induction of Th1 memory by RNA pTNT-Cyp c1.

  FIG. 27 shows the induction of Th1 memory by RNA pTNT-Fel d1.

  FIG. 28 shows the induction of Th1 memory by RNA pTNT-Der p2 and the suppression of IgE response.

  FIG. 29 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Alt a1.

  FIG. 30 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Cla h8.

  FIG. 31 shows the induction of Th1 memory by RNA pTNT-Hev b6.

  FIG. 32 shows the induction of Th1 memory by RNA pTNT-hybrid (allergen).

  FIG. 33 shows induction of Th1 memory and suppression of IgE response by RNA pTNT-Ara h2.

  FIG. 34 shows the induction of Th1 memory by RNA pTNT-Que a1.

  FIG. 35 shows the induction of Th1 memory by RNA pTNT-Art v1.

  FIG. 36 shows no induction of Th1 memory by RNA pTNT-Ole e1 or no suppression of IgE response.

<Example>
[Example 1:]
In this example, we show that replicase-based RNA vaccines encoding RNA and the clinically relevant millet moth pollen allergen Phl p5 effectively suppress allergic reactions.

(Materials and methods)
[Plasmid used for RNA transcription]
The pTNT vector was purchased from Promega (Mannheim, Germany). This vector has special features with advantages over other vectors. There are two promoters, one for SP6 polymerase and the other for T7 polymerase, allowing in vitro transcription based on SP6 and T7. These promoters are arranged in the vicinity of the multicloning site (MCS) in tandem. The A5'β-globin leader sequence facilitates increased translation of multiple genes by initiating translation more quickly. The vector has a synthetic poly (A) 30 tail as another feature that improves gene expression.

  The vector pSin-Rep5 (Invitrogen, Austria) is derived from Sindbis alphavirus, which is a wrapped positive strand RNA virus. Alphavirus-based replicon vectors lack the viral structural proteins but retain the replication elements (replicases) necessary for RNA self-propagation in the cytoplasm and expression of the inserted gene via the alphaviral promoter.

  The Phl p5 gene was extracted from the vector pCMV-Phlp5 via NheI / XbaI (Gabler et al. (2006), J Allergy Clin Immunol 118: 734-741) and linked to the XbaI restriction enzyme recognition sites of pTNT and pSin-Rep5. PTNT-P5 and pSin-Rep5-P5 were obtained respectively.

[RNA transcription]
Plasmids pTNT-P5 and pSin-Rep5-P5 were linearized using the corresponding restriction enzymes. The template was purified by phenol-chloroform-isoamyl alcohol extraction followed by one chloroform-isoamyl alcohol extraction. After adding 1/10 volume of 3M sodium acetate pH 5.2, the plasmid was precipitated with 2 volumes of 100% EtOH and washed 3 times with 70% EtOH.

All transcription reactions were performed using T7 or SP6 RiboMAX ^™ Large Scale RNA Production Systems (Promega) according to the manufacturer's protocol. Briefly, as a 100 μl reaction, 20 μl of transcription buffer, 30 μl of rNTP, 5 to 10 μg of template, and 10 μl of enzyme mixture were filled with nuclease-free H ₂ O to make 100 μl, and at 37 ° C. Incubated for ~ 3 hours. When using the SP6 RiboMax kit, 20 μl of rNTP was used instead of 30 μl of rNTP.

  In order to mimic the cap structure of mRNA, 5'7-methylguanosine nucleotides (m7G (5 ') ppp (5') G) or cap analogs (EPICENTRE, USA) were incorporated during RNA synthesis. The rNTP mixture was prepared to be a 25: 25: 25: 22.5: 2.5 mM mixture of rATP, rCTP, rUTP, rGTP and m7G (5 ') ppp (5') G.

After transcription, 1 volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation (13000 rpm) at 4 ° C. or room temperature for 15 minutes, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

(result)
[In vitro transfection using RNA and self-replicating RNA]
BHK-21 cells were transfected in vitro with two different RNA transcripts encoding β-galactosidase. The RNA transcript is a conventional RNA vaccine (βGal-RNA) transcribed from the vector pTNT-βGal or a self-replicating RNA (βGal-repRNA) transcribed from the vector pRep5-βGal.

  RNA transcripts with or without the m7G (5 ') ppp (5') G cap structure were tested. FIG. 1 shows that when transfected with the same amount of self-replicating RNA as conventional RNA, it induces 7.5-fold higher gene expression than conventional RNA. In addition, stabilization of RNA by the cap structure is essential for in vitro transfection / translation of RNA.

[RNA-based vaccine encoding allergen Phlp5 is immunogenic and suppresses IgE induction]
In order to verify the potential of RNA-based vaccines to suppress allergic induction, conventional RNA encoding Phl p 5 or self-replicating RNA encoding Phl P 5 was used in female BALB / c mice. Immunity was conferred. To calculate the effectiveness of RNA vaccines, the same amount of conventional DNA vaccine (pCMV-P5) and Phl p5 encoding self-replicating DNA vaccine (pSin-P5) were used to confer immunity to the corresponding groups . The mice were immunized three times at weekly intervals, and two weeks later, the recombinant Phl p 5 complexed with alum was injected twice to sensitize. This is known as a protocol for inducing an allergic phenotype and is characterized by high levels of IgE and a cytokine profile biased at TH2.

  FIG. 2A shows that both RNA vaccines induce a humoral immune response similar to the self-replicating DNA vaccine pSin-P5. In contrast, the humoral immune response induced by the conventional DNA vaccine pCMV-P5 was about an order of magnitude higher than other vaccines. All vaccine types showed a TH1 bias serological profile characterized by a low IgG1 / IgG2a ratio and did not show functional IgE induction as measured by the RBL release assay (FIG. 3, gray bar graph) ).

  After sensitization, the non-preimmunized control group showed fully TH2-biased serum with high IgG1 levels and high IgG1 / IgG2a ratio, indicating allergic sensitization. In contrast, all vaccinated groups maintained a TH1-harmonic immune phenotype (FIG. 2B). Pre-vaccination with both types of RNA vaccine resulted in the same or better suppression of IgE induction as the control animals with the corresponding DNA (Figure 3, black bar graph). Overall, pre-vaccination with both types of RNA vaccines suppressed IgE induction against allergic sensitization by 93%.

[RNA-based vaccine induces TH1-biased T cell memory]
Two weeks after the final sensitization, splenocytes were restimulated in vitro with recombinant Phl p 5 protein and their TH1 / TH2 profiles were evaluated. Therefore, the number of IFN-γ, IL-4, and IL-5 secreting cells was measured using ELISPOT.

  All the inoculated groups pre-vaccinated with the nucleic acid vaccine were shown to have significantly more induction of IFN-γ secreting cells than the control group (FIG. 4A). At the same time, the number of cells secreting the TH2-type cytokines IL-4 (FIG. 4B) and IL-5 (FIG. 4C) is suppressed and, like DNA vaccines, RNA vaccines can establish TH1 bias antigen-specific memory. It has been shown that this TH1 bias antigen-specific memory can be reactivated by subsequent allergen exposure.

[RNA-based vaccine reduces allergen-induced lung inflammation]
To examine the effect of RNA inoculation on the induction of pulmonary pathology, 2 μg of final sensitization, 1 μg of recombinant Phl p 5 was administered intranasally twice daily to induce pulmonary inflammation. The protocol strongly induced leukocyte infiltration into bronchoalveolar lavage fluid (BALF) in sensitized mice (FIG. 5A, control). Approximately 80% of the infiltrated leukocytes were eosinophils (FIG. 5B). In contrast, the total number of infiltrating leukocytes in the pre-vaccinated mice was significantly reduced, and further reduced for eosinophils.

  Inflammatory infiltration was also strongly influenced by the suppression of IL-5 in BALF and was reduced (FIG. 6A). Inhibition of IL-5 was inversely correlated with induction of IFN-γ (FIG. 6B).

(Conclusion)
DNA vaccines are very likely to be effective in preventing and treating allergic diseases. However, since there is a hypothesis that DNA vaccines are at risk, the clinical use of this new vaccine for healthy adults or children has been questioned.

  In this example, it was shown for the first time that inoculation with naked RNA having a clinically relevant allergen can suppress allergy induction to the same extent as when the same amount of DNA vaccine as this naked RNA was administered. .

  To address the problem of producing larger amounts of RNA, conventional RNA was compared with self-replicating RNA from the Sindbis virus replicon. In vitro transfection with both types of RNA, antigen expression was found to depend inter alia on the addition of the m7G (5 ') ppp (5') G cap analogologon. It is well known that most eukaryotic mRNAs have the m7G (5 ′) ppp (5 ′) G cap structure at the 5 ′ end, which is important for binding of translation initiation factors and contributes to mRNA stability. To do. In addition, a 7-fold higher level of protein was translated from a similar amount of self-replicating RNA (FIG. 1). This result contributes to the self-growth of subgenomic RNAs encoding the respective antigens. The result is the effect of contributing to the induction of apoptosis in the transfected cells, in contrast to self-replicating DNA vaccines, which have lower protein expression than conventional DNA vaccines. However, RNA vaccine expression is only transient and is therefore comparable to cells that undergo apoptosis immediately after being transfected with a self-replicating vaccine. In fact, self-replicating RNA vaccines elicit the same humoral immune response as self-replicating DNA vaccines (FIG. 2A), whereas conventional DNA vaccines continuously express the antigen and therefore have the highest humoral immune response. Show.

  In this example, a self-replicating nucleic acid vaccine of one fifth of the conventional RNA / DNA vaccine was used, but the increase in IgG2a after the next sensitization with recombinant allergen in alum (FIG. 2B), and Similar TH1 memory induction was observed as shown in the TH1 cytokine profile of restimulated splenocytes, as well as the high ability to suppress (FIG. 3). Thus, both RNA vaccines and self-replicating DNA vaccines show a higher suppressive capacity than conventional DNA vaccines. However, the latter induces higher levels of normal antigens and higher humoral immune responses. This indicates that vaccines that induce long-term allergen secretion can have adverse effects compared to short-term vaccine expression found in RNA and self-replicating vaccines.

  RNA vaccination also reduces lung infiltration after induction in the nasal cavity to the same extent as DNA vaccine (FIG. 5A). This is mainly due to the drastic decrease in the number of eosinophils in BALF (FIG. 5B). This correlates with the reduction of IL-5 in the lung (FIG. 6A) and the induction of intermediate levels of IFN-γ (FIG. 2B), where the production of TH1 cells induced by the vaccine indicates that TH1 / It has been shown to affect TH2 cytokine balance. In a viral model, IFN-γ in the lung has a detrimental effect on asthma and lung pathology, because IFN-γ activates lung epithelial cells and incorporates more TH2 cells into the tissue. It is considered an indirect effect. Indeed, in an allergy model, redirecting TH2 immunity to a more harmonious TH1 environment has a positive impact on lung infiltration and airway hypersensitivity, primarily by counter-regulating IL-5 and IL-13. (Ford, JG et al. (2001) J Immunol 167: 1769-1777).

  In summary, RNA-based vaccines can strongly induce protection against allergic sensitization, and the effects can be obtained at low doses by using self-replicating RNA vaccines. In view of this excellent safety profile in RNA vaccines, there is a possibility that RNA vaccines can be used not only in therapeutic environments but also in healthy individuals who are likely to develop allergic diseases.

[Example 2]
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Bet v 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol. Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation at 4 ° C. or room temperature for 15 minutes, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Bet v 1 three times at weekly intervals. One week later, 1 μg recombinant Bet v 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Bet v 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-, an indicator of allergen-specific Th1 cell activation. Analyzed for γ.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Bet v 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 7A) and increased secretion of IFN-γ (FIG. 7B) resulted in an allergen specific Th1 cell recruitment. The first stimulation of Th1 was able to suppress the induction of allergen-specific IgE response (FIG. 7C).

Example 3
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Car b 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

The capped transcript was incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation at 4 ° C. or room temperature for 15 minutes, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Car b 1 three times at weekly intervals. One week later, 1 μg of recombinant Car b 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Car b 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-, an indicator of allergen-specific Th1 cell activation. Analyzed for γ.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Car b 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 8A) and increased secretion of IFN-γ (FIG. 8B), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 8C).

Example 4
(Materials and methods)
[Plasmid and RNA transcription]
The cDNA encoding Cas s 1 was cloned into the vector pTNT as described in Example 1. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation (13000 rpm) for 15 minutes at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Cas s 1 three times at weekly intervals. One week later, 1 μg of recombinant Cass 1 in a complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Cas s 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-, an indicator of allergen-specific Th1 cell activation. Analyzed for γ.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Cas s 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 9A) and increased secretion of IFN-γ (FIG. 9B), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 9C).

Example 5
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Phl p 1 was cloned into the vector pTNT. RNA transcripts were generated as described above and cap-like structures were added using ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation (13000 rpm) for 15 minutes at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Phl p 1 three times at weekly intervals. One week later, 1 μg of recombinant Phl p 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a was measured using ELISA. Ten days after the final sensitization, spleen cells were restimulated with recombinant Phl p 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-, an indicator of allergen-specific Th1 cell activation. Analyzed for γ.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 10A) and increased secretion of IFN-γ (FIG. 10B) resulted in an allergen-specific Th1 cell recruitment.

Example 6
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Phl p 6 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

The capped transcript was incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After centrifugation (13000 rpm) for 15 minutes at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Phl p 6 three times at weekly intervals. One week later, 1 μg of recombinant Phl p 6 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense>]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Phl p 6 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-, an indicator of allergen-specific Th1 cell activation. Analyzed for γ.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 6 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 11A) and increased secretion of IFN-γ (FIG. 11B), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 11C).

Example 7
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Cor a 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized three times at weekly intervals using RNA pTNT-Cor a1. One week later, 1 μg of recombinant Cor a 1 in a complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgG2a was measured using ELISA.

(result)
In contrast to sensitized controls (black bar graph) or untreated mice (white bar graph), pre-vaccination with RNApTNT-Cor a 1 (hatched bar graph) increased the induction of IgG2a (FIG. 12A). ) And increased secretion of IFN-γ (FIG. 12B) resulted in allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 12C).

Example 8
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Aln g 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

The capped transcript was incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Aln g 1 three times at weekly intervals. One week later, 1 μg of recombinant Alng 1 complexed with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
Ten days after the final sensitization, spleen cells were restimulated with recombinant Aln g 1 for 72 hours in vitro, and the cell culture supernatant was assayed for IFN-γ, an indicator of allergen-specific Th1 cell activation. analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Aln g 1 (hatched bar graphs) shows IFN-γ secretion. It resulted in an allergen-specific Th1 cell recruitment as shown by the increase (FIG. 13).

Example 9
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Fags 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Fag s 1 three times at weekly intervals. One week later, 1 μg of recombinant Fags 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Fags 1 for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Fag s 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 14A) and increased secretion of IFN-γ (FIG. 14B), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 14C).

Example 10
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Phl p 2 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Phl p 2 three times at weekly intervals. One week later, 1 μg recombinant Phl p 2 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgE was measured using RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Phl p 2 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-γ, an indicator of activation of allergen-specific Th1 cells. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 2 (hatched bar graphs) shows the secretion of IFN-γ. As shown by the increase (FIG. 15A), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE response (FIG. 15B).

[Embodiment 11]
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Phl p 7 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

The capped transcript was incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Phl p 7 three times at weekly intervals. One week later, 1 μg of recombinant Phl p 7 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgE was measured using RBL as described in Experiment 1.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 7 (hatched bar graphs) shows IFN-γ secretion. As shown by the increase (FIG. 16A), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 16B).

Example 12
(Materials and methods)
[Plasmid and RNA transcription]
Hybrid cDNAs encoding Phl p 1, Phl p 2, Phl p 5, and Phl p 6 were cloned into the vector pTNT as described in Example 1 (Linhart B. and Valenta R., Int Arch). Allergy Immunol (2004) 134: 324-331). RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized three times at weekly intervals with RNA pTNT-hybrid (Phl p 1-2-5-6). One week later, 1 μg of recombinant Phl p 1, Phl p 2, Phl p 5, and Phl p 6 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after final sensitization, spleen cells were restimulated with recombinant allergens in vitro for 72 hours, and cell culture supernatants were analyzed for IFN-γ, an indicator of allergen-specific Th1 cell activation. did.

(result)
Pre-vaccine with RNA pTNT-hybrid (Phl p 1-2-5-6) (shaded bar graph), in contrast to sensitized controls (black bar graph) or untreated mice (white bar graph) Inoculation resulted in a gradual increase in allergen-specific Th1 cells, as shown by increased induction of IgG2a (FIG. 17A) and increased secretion of IFN-γ (FIG. 17B). The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 17C).

Example 13
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Cry j 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Cry j 1 three times at weekly intervals. One week later, 1 μg of recombinant Cry j 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a was measured using ELISA. Ten days after the final sensitization, spleen cells were restimulated with recombinant Cry j 1 for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Cry j 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 18A) and increased secretion of IFN-γ (FIG. 18B), resulted in an allergen-specific Th1 cell recruitment.

Example 14
(Materials and methods)
[Plasmid and RNA transcription]
The cDNA encoding Jun a 1 was cloned into the vector pTNT as described in Example 1. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Jun a 1 3 times at weekly intervals. One week later, 1 μg of recombinant Jun a 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
Ten days after the final sensitization, spleen cells were restimulated with recombinant Jun a 1 for 72 hours in vitro, and the cell culture supernatant was used as an indicator of activation of allergen-specific Th1 cells, IFN-γ. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Jun a 1 (hatched bar graphs) shows IFN-γ secretion. It resulted in an allergen-specific Th1 cell recruitment as shown by the increase (FIG. 19).

Example 15
(Materials and methods)
[Plasmid and RNA transcription]
Cloning the cDNA encoding Amb a 1 into the vector pTNT as described in Example 1. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Amb a1 three times at weekly intervals. One week later, 1 μg of recombinant Amb a1 complexed with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
Ten days after the final sensitization, spleen cells were restimulated with recombinant Amb a 1 for 72 hours in vitro, and the cell culture supernatant was used as an indicator of activation of allergen-specific Th1 cells, IFN-γ. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Amb a 1 (hatched bar graphs) shows the secretion of IFN-γ. As shown by the increase (FIG. 20), it resulted in an allergen-specific Th1 cell recruitment.

Example 16
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Apig 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.
Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Api g 1 three times at weekly intervals. One week later, 1 μg of recombinant Apig 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Api g 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 6 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 21A) and increased secretion of IFN-γ (FIG. 21B), resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 21C).

Example 17
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Dau c 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Dau c 1 three times at weekly intervals. One week later, 1 μg of recombinant Dau c1 complexed with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a was measured using ELISA. Ten days after the final sensitization, spleen cells were restimulated with recombinant Dau c 1 for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Phl p 6 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 22A) and increased secretion of IFN-γ (FIG. 22B) resulted in an allergen-specific Th1 cell recruitment.

Example 18
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Mal d 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Mal d 1 3 times at weekly intervals. One week later, 1 μg of recombinant Mal d 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, the splenocytes were restimulated with recombinant Mal d1 for 72 hours in vitro, and the cell culture supernatant was assayed for IFN-γ, an indicator of allergen-specific Th1 cell activation. analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Mal d 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 23A) and increased secretion of IFN-γ (FIG. 23B) resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 23C).

Example 19
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Ova was cloned into the vector pTNT. RNA transcripts were generated as described above and cap-like structures were added using ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Ova three times at weekly intervals. One week later, 1 μg of recombinant Ova complexed with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after final sensitization, spleen cells were restimulated with recombinant Ova for 72 hours in vitro, and cell culture supernatants were analyzed for IFN-γ, an indicator of allergen-specific Th1 cell activation. did.

(result)
In contrast to sensitized controls (black bar graph) or untreated mice (white bar graph), pre-vaccination with RNA pTNT-Ova (hatched bar graph) increased the induction of IgG2a (FIG. 24A). ) And increased secretion of IFN-γ (FIG. 24B) resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 24C).

Example 20
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding beta-casein was cloned into the vector pTNT. RNA transcripts were generated as described above, and cap-like structures were added using ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-beta-casein three times at weekly intervals. One week later, 1 μg of recombinant beta-casein in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgE was measured using RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant beta-casein for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-beta-casein (hatched bar graphs) shows IFN-γ secretion. As shown by the increase (FIG. 25A), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 25B).

Example 21
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Cyp c 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with RNA pTNT-Cyp c 1 three times at weekly intervals. One week later, 1 μg of recombinant Cyp c 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a was measured using ELISA.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Cyp c 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 26), allergen-specific Th1 cell recruitment resulted.

[Example 22]
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Fel d 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Fel d 1 three times at weekly intervals. One week later, 1 μg of recombinant Fel d 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgG2a was measured using ELISA as described in Experiment 1. Ten days after the final sensitization, spleen cells were restimulated with recombinant Fel d 1 for 72 hours in vitro, and the cell culture supernatant was recovered from IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Fel d 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 27A) and increased secretion of IFN-γ (FIG. 27B), resulted in an allergen-specific Th1 cell recruitment.

Example 23
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Der p 2 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Der p 2 three times at weekly intervals. One week later, 1 μg of recombinant Der p 2 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Der p 2 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 11A), allergen-specific Th1 cell recruitment resulted. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 28B).

Example 24
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Alt a 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Alt a 1 three times at weekly intervals. One week later, 1 μg of recombinant Alt a 1 using alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Alt a 1 for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Alt a 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 29A) and increased secretion of IFN-γ (FIG. 29B), resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 29C).

Example 25
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Cla h 8 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Cla h 8 three times at weekly intervals. One week later, 1 μg of recombinant Cla h 8 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgE was measured using RBL as described in Experiment 1. Ten days after the final sensitization, spleen cells were restimulated with recombinant Cla h 8 for 72 hours in vitro, and the cell culture supernatant was assayed for IFN-γ, an indicator of allergen-specific Th1 cell activation. analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Cla h 8 (hatched bar graphs) shows IFN-γ secretion. As shown by the increase (FIG. 30A), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 30B).

Example 26
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Hev b 6 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Hev b 6 three times at weekly intervals. One week later, 1 μg of recombinant Hev b 6 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgG2a was measured using ELISA as described in Experiment 1. Ten days after the final sensitization, spleen cells were restimulated with recombinant Hev b 6 for 72 hours in vitro, and the cell culture supernatant was used as an indicator of activation of allergen-specific Th1 cells, IFN-γ. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Hev b 6 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 31A) and increased secretion of IFN-γ (FIG. 31B), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of an allergen-specific IgE response (FIG. 31C).

Example 27
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, hybrid cDNAs encoding 5 different allergens were cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized three times at weekly intervals using RNA pTNT-hybrid (Aln-Cor-Que-Car-Bet). One week later, 1 μg of recombinant allergen in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgG2a was measured using ELISA as described in Experiment 1.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-hybrid (hatched bar graph) increased the induction of IgG2a. As shown by (FIG. 32), it resulted in an allergen-specific Th1 cell recruitment.

Example 28
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Ara h 2 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.
Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Ara h 2 three times at weekly intervals. One week later, 1 μg of recombinant Ara h 2 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Ara h2 for 72 hours in vitro and the cell culture supernatant was analyzed for IFN-γ as an indicator of allergen-specific Th1 cell activity.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Ara h 2 (hatched bar graphs) shows IFN-γ secretion. As shown by the increase (FIG. 33A), it resulted in an allergen-specific Th1 cell recruitment. The initial stimulation of Th1 was able to suppress the induction of allergen-specific IgE responses (FIG. 33B).

Example 29
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, the cDNA encoding Queue a 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized three times at weekly intervals with RNA pTNT-Quea1. One week later, 1 μg of recombinant Que a 1 complexed with alum was injected for 2 weeks and sensitized 1 week later to induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, allergen-specific serum IgG2a was measured using ELISA as described in Experiment 1. Ten days after the final sensitization, spleen cells were restimulated with recombinant Phl p 6 for 72 hours in vitro, and the cell culture supernatant was treated with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
In contrast to sensitized controls (black bar graphs) or untreated mice (white bar graphs), pre-vaccination with RNA pTNT-Queue a 1 (hatched bar graph) increased the induction of IgG2a ( As shown by FIG. 34A) and increased secretion of IFN-γ (FIG. 34B) resulted in an allergen-specific Th1 cell recruitment.

Example 30
(Materials and methods)
[Plasmid and RNA transcription]
The cDNA encoding Art v 1 was cloned into the vector pTNT as described in Example 1. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Art v 1 three times at weekly intervals. One week later, 1 μg of recombinant Art v 1 in complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a was measured using ELISA and RBL. Ten days after the final sensitization, spleen cells were restimulated with recombinant Art v 1 for 72 hours in vitro, and the cell culture supernatant was reconstituted with IFN-γ, an indicator of allergen-specific Th1 cell activation. Was analyzed.

(result)
Pre-vaccination with RNA pTNT-Art v 1 (hatched bar graph) is shown by no increase in IgG2a induction (FIG. 35A) and no increase in IFN-γ secretion (FIG. 35B). As can be seen, there was no allergen-specific Th1 cell recruitment.

[Example 31]
(Materials and methods)
[Plasmid and RNA transcription]
As described in Example 1, cDNA encoding Ole e 1 was cloned into the vector pTNT. RNA transcripts were prepared as described above and cap-like structures were added using a ScriptCap kit (Ambion) according to the manufacturer's protocol.

Capped transcripts were incubated with RNAse-free DNAse (Promega) for 15 minutes at 37 ° C. to remove template DNA. One volume of 5M ammonium acetate was added to the reaction tube and the mixture was incubated on ice for 10-15 minutes to precipitate the RNA. After 15 minutes centrifugation (13000 rpm) at 4 ° C. or room temperature, the precipitate was washed with 70% ethanol and resuspended in nuclease-free H ₂ O.

[Immunization and sensitization]
Mice were immunized with the RNA pTNT-Ole e 1 three times at weekly intervals. One week later, 1 μg of recombinant Ole e 1 in a complex with alum was injected for 2 weeks to sensitize and induce an allergic phenotype. Control animals were sensitized only and not pre-vaccinated with RNA vaccines.

[Measure Th1 memory induction and defense]
One week after the final sensitization, as described in Experiment 1, allergen-specific serum IgG2a and IgE were measured using ELISA and RBL.

(result)
Pre-vaccination with RNA pTNT-Ole e 1 (hatched bar graph) is shown by no increase in IgG2a induction (FIG. 36A) and no increase in IFN-γ secretion (FIG. 36B). As can be seen, there was no allergen-specific Th1 cell recruitment. Furthermore, inhibition of induction of allergen-specific IgE reaction could not be measured (FIG. 36B).

FIG. 6 shows in vitro transfection of BHK21 cells using RNA encoding β-galactosidase (βGal-RNA) or self-replicating RNA (βGal-repRNA) transcripts. RNA transcripts (cap) with or without the m7G (5 ') ppp (5') G cap structure were tested (no cap). Untransfected cells were used as a background control (not transfected). Data are shown as the mean ± standard error (SEM) of three independent transfection experiments. Shown are Phl p 5-specific IgG1 and IgG2a levels after nucleic acid vaccination. Serum was diluted 1: 1000 (FIG. 2A). The numerical value on the top of the bar graph indicates the average ratio of IgG1: IgG2 in each group. Data represent mean ± SEM (n = 4). Shows subsequent sensitization with recombinant allergens in alum. Serum was diluted 1: 100,000 (FIG. 2B). The numerical value on the top of the bar graph indicates the average ratio of IgG1: IgG2 in each group. Data represent mean ± SEM (n = 4). 2 shows Phl p 5 specific IgE measured by RBL release assay. IgE levels were measured after vaccination with each nucleic acid vaccine (grey bar graph) and after subsequent sensitization with recombinant allergen in alum (black bar graph). Numbers represent the mean ± SEM (n = 4) of specific hexosaminidase release. ***: P <0.001. FIG. 4 shows the number of IFN-gamma (FIG. 4A) after in vitro re-stimulation with recombinant Phl p 5 as measured using ELISPOT. Data show mean ± SEM (n = 4) of the number of cytokine secreting cells per 10 ⁶ splenocytes. FIG. 4 shows the number of IL-4 (FIG. 4B) after in vitro restimulation with recombinant PhI p 5 measured using ELISPOT. Data show mean ± SEM (n = 4) of the number of cytokine secreting cells per 10 ⁶ splenocytes. FIG. 4 shows the number of IL-5 (FIG. 4C) after in vitro re-stimulation with recombinant Phl p 5 measured using ELISPOT. Data show mean ± SEM (n = 4) of the number of cytokine secreting cells per 10 ⁶ splenocytes. FIG. 5 shows the total white blood cell count in BALF of sensitized mice after intranasal administration of allergen (FIG. 5A). A numerical value shows average value +/- SEM (n = 4). *: P <0.05; **: P <0.01. FIG. 5 shows eosinophil counts in BALF of sensitized mice after intranasal administration of allergen (FIG. 5B). A numerical value shows average value +/- SEM (n = 4). *: P <0.05; **: P <0.01. FIG. 6 shows IL-5 (FIG. 6A) levels in BALF of sensitized mice after intranasal administration of allergens. Data show mean ± SEM (n = 4). *: P <0.05; **: P <0.01; ***: P <0.001. FIG. 6 shows the levels of IFN-γ (FIG. 6B) in BALF of sensitized mice after intranasal administration of allergens. Data show mean ± SEM (n = 4). *: P <0.05; **: P <0.01; ***: P <0.001. Induction of Th1 memory and suppression of IgE response by RNA pTNT-Bet v1. Induction of Th1 memory by RNA pTNT-Bet v 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Bet v 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Car b 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Car b 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Car b 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cas s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cas s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cas s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 1 is shown. Induction of Th1 memory by RNA pTNT-Phl p 1 is shown. Induction of Th1 memory by RNA pTNT-Phl p 6 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 6 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 6 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cor a 1 is shown. Induction of Th1 memory by RNA pTNT-Aln g 1 is shown. Induction of Th1 memory by RNA pTNT-Fag s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Fag s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Fag s 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 7 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Phl p 7 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-hybrid (Phl p1-2-5-6) and suppression of IgE response. Induction of Th1 memory by RNA pTNT-hybrid (Phl p1-2-5-6) and suppression of IgE response. Induction of Th1 memory by RNA pTNT-hybrid (Phl p1-2-5-6) and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cry j 1 is shown. Induction of Th1 memory by RNA pTNT-Cry j 1 is shown. Induction of Th1 memory by RNA pTNT-Jun a 1. Induction of Th1 memory by RNA pTNT-Amb a 1 is shown. Induction of Th1 memory by RNA pTNT-Api g 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Api g 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Api g 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Dau c 1 is shown. Induction of Th1 memory by RNA pTNT-Dau c 1 is shown. Induction of Th1 memory by RNA pTNT-Mal d 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Mal d 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Mal d 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Ova and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Ova and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Ova and suppression of IgE response. The induction | guidance | derivation of Th1 memory by RNA pTNT-Beta-Casein and suppression of IgE reaction are shown. The induction | guidance | derivation of Th1 memory by RNA pTNT-Beta-Casein and suppression of IgE reaction are shown. Induction of Th1 memory by RNA pTNT-Cyp c 1 is shown. Induction of Th1 memory by RNA pTNT-Fel d 1 is shown. Induction of Th1 memory by RNA pTNT-Fel d 1 is shown. Induction of Th1 memory by RNA pTNT-Der p 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Der p 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Alt a 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Alt a 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Alt a 1 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cla h 8 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Cla h 8 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Hev b 6 is shown. Induction of Th1 memory by RNA pTNT-Hev b 6 is shown. Induction of Th1 memory by RNA pTNT-hybrid (allergen). Induction of Th1 memory by RNA pTNT-Ara h 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Ara h 2 and suppression of IgE response. Induction of Th1 memory by RNA pTNT-Queue a 1 is shown. Induction of Th1 memory by RNA pTNT-Queue a 1 is shown. Induction of Th1 memory by RNA pTNT-Art v 1 is shown. Induction of Th1 memory by RNA pTNT-Art v 1 is shown. It shows that there is no induction of Th1 memory by RNA pTNT-Ole e 1 or no inhibition of IgE reaction. It shows that there is no induction of Th1 memory by RNA pTNT-Ole e 1 or no inhibition of IgE reaction.

Claims (16)

  An RNA vaccine comprising at least one RNA molecule encoding at least one allergen or a derivative thereof, wherein the allergen is Alnus glutinosa, Alternaria alternata, Ambrosia artemisifolia (Ambrosia) artemisiifolia, Apium graveolens, Arachis hypogaea, Betula verrucosa, Carpinus betulus, Castanea sativa sp, Helbarum poly herbarum, Corylus avellana, Cryptomeria japonica, Cyprinus carpio, Daucus carota, Delmatofagoide Pteronysinus (Dermatophagoides pteronyssinus), Fagus sylvatica (Fagus sylvatica), Ferris domesticus (Felis domesticus), Hevea brasiliensis, Juniperus ashei (Juniperus ashei), Malus domestica An RNA vaccine, which is an allergen of Alba (Quercus alba) and Phleum pratense. The allergen of Arnus glutinosa is Aln g 1, and the allergen of Altanaria alternata is Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8, The allergen selected from the group consisting of Alt a 10, Alt a 12, and Alt a 13, and the allergen of Ambrosia artemisifolia is Amb a 1, Amb a 2, Amb a 3, Amb a 5, Amb a 6, Amb a 7, selected from the group consisting of Amb a 8, Amb a 9 and Amb a 10, wherein the allergen of Apium gravelens is selected from the group consisting of Api g 1, Api g 4 and Api g 5, and from Arakis Hypogaea The allergens are Ara h 1, Ara h 2, Ara h 3 , Ara h 4, Ara h 5, Ara h 6, Ara h 7, and Ara h 8, and the allergens of Betella velocosa are Bet v 1, Bet v 2, Bet v 3, Bet v 4 , Bet v 6 and Bet v 7 and the allergen of Carpinus betulus is Car b 1, and the allergen of Castanea sativa consists of Cas s 1, Cas s 5 and Cas s 8 The allergen of Cladosporium herbalum selected from the group is selected from the group consisting of Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9, Cla h 10, and Cla h 12. The selected allergens of Corylus averana are Cor a 1, Cor a 2, Cor a 8, C selected from the group consisting of or a 9, Cor a 10 and Cor a 11, wherein the allergen of Cryptomeria japonica is selected from the group consisting of Cry j 1 and Cry j 2, and the allergen of Caprinas Carpio is Cyp c 1 and the allergen of Daux-Carota is selected from the group consisting of Dau c 1 and Dau c 4, and the allergen of Dermatophagoides pteronisinus is Der p 1, Der p 2, Der p 3, from der p 4, der p 5, der p 6, der p 7, der p 8, der p 9, der p 10, der p 11, der p 14, der p 20, der p 21 and Clone 3 0 allergens The allergen of Fagus Silvatica is selected from the group consisting of: s 1 and the allergen of Ferris domesticus is selected from the group consisting of Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 5w, Fel d 6w and Fel d 7w, and Hevea brush The above allergens of Reensis are Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6.01, Hev b 6.02, Hev b 6.03, Hev b 7.01. , Hev b 7.02, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, and Hev b 13, and the allergen of Juniperus Ashey is Jun a 1, Jun selected from the group consisting of a 2 and Jun a 3, and the above allergen of Mars Domestica is Mal d 1, M selected from the group consisting of l d 2, Mal d 3 and Mal d 4, the allergen of Kercus alba is Que a 1 and the allergen of Fleum platen is Phl p 1, Phl p 2, The vaccine according to claim 1, wherein the vaccine is selected from the group consisting of Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12, and Phl p 13. The allergen, Aln g 1, Alt a 1 , Amb a 1, Api g 1, Ara h 2, Bet v 1, C ar b 1, Cas s 1, Cla h 8, Cor a 1, Cry j 1, Cyp c 1, Dau c 1, Der p 2, Fag s 1, Fel d 1, Hev b 6, Jun a 1, Mal d 1, P hl p 1, Phl p 2, Phl p 5, Phl p 6 and Phl p The vaccine according to claim 1 or 2, wherein the vaccine is selected from the group consisting of 7.   The RNA molecule encodes Phl p 1, Phl p 2, Phl p 5 and Phl p 6, or Aln g 1, Cor a 1, Que a 1, Car b 1, and Bet v 1 Item 3. The vaccine according to Item 1 or 2.   The vaccine according to any one of claims 1 to 4, wherein the allergen derivative is hypoallergenic.   The IgE reactivity of the hypoallergenic allergen derivative is 10% or more, preferably 20% or more, more preferably 30% or more, particularly 50% or more lower than the IgE reactivity of the wild type allergen The vaccine according to claim 5.   7. The RNA molecule encoding the allergen or a derivative thereof is bound to at least one further molecule encoding a peptide, polypeptide or protein according to any one of claims 1-6. vaccine.   8. The RNA molecule according to any one of claims 1 to 7, characterized in that said RNA molecule comprises at least one further element selected from the group consisting of replicase, [beta] -globin leader sequence, cap0, cap1 and poly A tail. The vaccine described. The vaccine, the vaccine according to any one of claims 1 to 8, wherein further including Ndei Rukoto the A adjuvant.   The vaccine according to claim 9, wherein the adjuvant is selected from the group consisting of CpG-DNA and cytokines, and the cytokines are preferably IL-12 and IL-15. The vaccine according to any one of claims 1 to 10 , wherein the vaccine is applied to intramuscular administration, intradermal administration, intravenous administration, transdermal administration, topical administration, or fine particle gun administration. vaccine.   Use of at least one RNA molecule according to any one of claims 1 to 8 for the manufacture of a vaccine for the treatment or prevention of allergies.   Use of at least one RNA molecule according to any one of claims 1 to 8 for the manufacture of a prophylactic and therapeutic vaccine for desensitization of an individual against an allergen. Said vaccine Use according to claim 12 or 13, characterized in further including Ndei Rukoto the A adjuvant.   Use according to claim 14, characterized in that the adjuvant is selected from the group consisting of CpG-DNA and cytokines, said cytokines being preferably IL-12 and IL-15. The use according to any one of claims 12 to 15 , wherein the vaccine is applied to intramuscular administration, intradermal administration, intravenous administration, transdermal administration, topical administration, or fine particle gun administration. .

Images